It is the conventional dogma that chymotrypsin is synthesized and stored in the exocrine pancreas and secreted into the small intestine for digestion of proteins. A current ongoing study utilizing a chymotrypsin antiserum has suggested to us that chymotrypsin may be synthesized in the brain. We therefore undertook a series of experiments to further explore the possibility that chymotrypsin may be synthesized in the brain and also within peripheral tissues. Rat pancreatic RNA was isolated in the presence of guanidine thiocyanate. Polyadenylated RNA was isolated from total pancreatic RNA by binding to oligo (dT)-cellulose. A 5' primer (27- mer), including an upstream EcoR l site, and a 3'-primer (28-mer), including a downstream Hind Ill site, were designed to amplify a portion of cDNA (about 500 pb) from rat pancreatic mRNA using PCR. The sequence of the fragment was confirmed to be a portion overlapping exon-6 and exon-7 of rat chymotrypsin B gene. It was subcloned into the pGEM-4Z vector and used as template for in vitro transcription of an antisense riboprobe. Using the RNase protection procedure developed in our laboratory for quantitation of mRNA in discrete micropunches of brain regions, we have detected chymotrypsin mRNA in rat brain. We found that (1) regions such as the hippocampus, striatum, thalamus and cortex contain chymotrypsin mRNA; (2) some peripheral organs, such as the ovary, spleen and stomach also contain chymotrypsin mRNA and appeared to be more concentrated in these organs compared to the brain; (3) further evidence that chymotrypsin mRNA exists in rat brain regions was provided by Northern blots. The presence of chymotrypsin in the brain is of particular interest in neurodegenerative diseases such as Alzheimer's. The presence of chymotrypsin in the stomach suggests that protein metabolism of foodstuff may take place prior to entry into the duodenum. We cannot yet begin to fathom the significance of chymotrypsin in the ovary and spleen.